Compositions and methods for improving the quality of processed sperm

ABSTRACT

The present invention generally relates to compositions and methods for the handling of processed sperm populations including samples that are freshly collected, those transported as fresh samples, as well as samples that are frozen and thawed, those sorted into one or more subpopulations, and those that are otherwise processed or handled that impose trauma on the sperm cell. Such trauma can reduce the motility, fertility, viability and overall integrity of the sperm and reduce the sperm&#39;s ability to fertilize an egg, grow into a health embryo and produce a healthy offspring. The present invention relates to novel compounds that can be added to the sperm cell sample to reduce the traumatic effects of physical stress during mild as well as extensive sperm cell processing, methods of using the compounds in standard sperm processing procedures, the end products made from these methods including sperm and embryos, as well as methods of using those end products in assisted reproductive biology techniques in animals

This application is a Continuation-in-Part of U.S. application Ser. No.13/844,636 filed Mar. 15, 2013, which is a Continuation of U.S.application Ser. No. 13/844,273 filed Mar. 15, 2013, which is aContinuation of the U.S. National Stage application Ser. No. 13/823,843filed Mar. 15, 2013, of International Application No. PCT/US2012/040553,filed Jun. 1, 2012, which claims priority to U.S. ProvisionalApplications 61/492,151 filed Jun. 1, 2012, 61/569,143 filed Dec. 9,2011, and 61/570,691 filed Dec. 14, 2011, the contents of which are allincorporated by reference in their entireties.

The present invention generally relates to compositions and methods forthe handling of processed sperm populations, including those freshlycollected as well as those sorted into one or more subpopulations, andfor treating processed and/or handled semen samples and solutions thatcontain sperm cells to increase the overall quality of the processedsperm, including their viability, motility, fertility, DNA integrity,and in vitro longevity. The present invention also relates tocompositions comprising sperm cells and at least one compound that maybe an antioxidant, vitamin or other organic stress reducer, the methodsof using these compounds to reduce trauma and stress on processed sperm,the resulting sperm and embryo end products, and the methods of use ofthese products in assisted reproductive technologies (ART) to increasethe quality, quantity and viability of embryos, and improved rates ofbirths in animals.

BACKGROUND

Assisted reproductive technology (ART) includes such techniques as invitro fertilization (IVF), artificial insemination (AI),intracytoplasmic sperm injection (ICSI) (other techniques usingenucleated cells) and multiple ovulation and embryo transfer (MOET) (aswell as other embryo transfer techniques), is used across the animalkingdom, including humans and other animals. ART methods are usuallyexpensive, time consuming and marginally successful given the inherentfragility of gametes and embryos when outside of their naturalenvironments. Furthermore, the use of ART within the animal breedingindustry in a commercially feasible manner is additionally challengingdue to the limited availability of genetically desirable gametes andzygotes. One way to lower the cost of ART and to improve its commercialfeasibility is to increase the efficiency of the involved processes byimproving the viability and overall quality of gametes and zygotes.Although there is has been a growing interest in this field over thecourse of the last decade or so, there still remains a strong need toincrease the overall quality of gametes and zygotes for use in ART,especially when breeding focuses on pre-natal gender selection,including improving their viability (in the case of gametes andzygotes), their motility and fertility (in the case of sperm cells), aswell as other longevity characteristics.

For example, in conventional AI, one problem limiting its commercialapplication in certain species is the need to use extremely high numberof sperm cells per AI dose to ensure successful fertilization.Similarly, in IVF, the percentage of zygotes that develop into embryosremains frustratingly low; this high rate of loss significantlyincreases the cost of embryos and related services to end-users. Therealso remains the need for more efficient and lower cost procedures forimproving post-embryo handling through cryopreservation as well asnon-frozen transport. Cryopreservation of embryos is limited by thesuccess rate of embryo production as well as blastocyst growth in vitro.Currently, only a marginal percentage of IVF embryos are suitable forcryopreservation which adds to the ongoing high cost of ART procedures.

Especially when processing gametes such as flushed oocytes or spermcells, both conventional and sex-sorted, before their use in ART adds atremendous amount of stress on the gamete cell and negatively impactstheir cellular integrity and membrane structure which in turn isreflected in decreased viability, motility and fertility. An example ofprocessing gametes prior to their use in ART is the sorting of spermcells based on sex (known as “gender enrichment” or “sex-sorting”),which is a highly desired procedure to minimize wasted births of thewrong sex for selective breeding in the livestock industry but is oftencost prohibitive and can be risky to those with smaller breeding herds.

The popular flow cytometry based sex-sorting process severely stressesand damages the cells and produces a low percentage of useful sperm,which although capable of fertilizing matured oocytes, have reducedviability, motility and fertility after the sex-sorting process.Typically, sex-sorting involves many harsh steps including but notlimited to: the initial collection and handling of sperm ejaculate whichnaturally starts to deteriorate rapidly upon collection; the staining ofsperm cells which involves binding of an excitable dye to the DNA or aharmful membrane selection procedure; the physical sorting of the spermcells using high energy fluorescence that physically energizes the dyethat is bound to the DNA, forced orientation through a narrow orifice,and application of an electrical charge to the cell; the physicalcollection of the cells into a container which often shocks the fragilecell upon contact; the osmotic stresses associated with dilution of thesperm droplet in collection media; and the storage of the sorted spermusually by freezing which is well known to raise havoc with the cell'smembrane systems. Each step places the processed sperm under abnormalstress which diminishes the overall motility, viability and/or fertilityof the sperm. The result can lead to less efficient samples for use inART, such as IVF and AI, and other types of subsequent or furtherprocessing.

Even non-sorted processed sperm exhibits significant losses infertility, viability and motility when being collected, handled andtransported without freezing, and noticeably experiences significantstress when mixed with cryoprotectant and is frozen and thawed. Many inthe field have tried to improve methods for the use on unsorted,conventional semen to minimize loss in the handling processes associatedwith in vitro handling, preservation and use of semen samples.

Regardless of the processing, sperm lose their potential to fertilizewhen exposed to: elevated temperatures, abnormal buffers, stains,altered pH systems, physical pressurized orientation as when forcedthrough a nozzle or when oscillated to form drops in a flow cytometer,radiation used to illuminate the DNA binding dye, physical stressorsassociated with separation and collection techniques, cryoprotectants,freezing, thawing and micromanipulation by the handler.

The large class of compounds referred to as antioxidants have beenassociated with providing beneficial effects to all sorts of cells, invivo and in vitro, but these effects are as varied as the nature of theantioxidant itself. An antioxidant is simply one of a large variety ofmolecules that either inhibit the oxidation of another molecule, becomesoxidized itself in place of the target substrate, or binds harmful freeradical intermediates and interrupts oxidative chain reactions within acell. Most have dual roles; some are enzymes, others are non-enzymatic;some others are vitamins and others are cofactors. Such diversity laudsthe diversity of antioxidants, but because of their known ability tominimize cell damage, they are frequently lumped together as a singleclass of compounds having only a single function, to bind free radicals.

Various antioxidants have shown promise in promoting cell integrity withsome reports showing positive effects on sperm motility and membraneintegrity during cryopreservation, but some tests have been shown tohave minimal or even harmful effects on processed sperm.

Similarly, vitamins are again a rather diverse group of molecules havingvery different biological properties. Vitamins are any of a large groupof organic compounds required in very small amounts as vital nutrientsfor an organism that cannot synthesize it. They can be antioxidants,enzymes, hormones or non-enzymes; they can be regulators of cell growth,cell differentiation or moderators of mineral metabolism.

To date, no studies have sufficiently addressed the use of antioxidants,vitamins or other supplements in the routine handling of fragile gametesduring in vitro processing, especially during the harsh processingassociated with the sex-sorting of sperm, whereby the end result is areproducible improvement on the viability, motility and fertility ofextensively processed sperm cells and embryos. There remains acontinuing need to improve current methods of ART to reduce the cost andto make the procedures more dependable and commercially feasible tothose on a tight budget, especially those smaller breeders who viewsex-selection breeding as a high risk and expensive option.

SUMMARY OF THE INVENTION

A broad object of the present invention is to provide improvements inthe motility, viability, fertility and overall integrity of processedsperm cells. Accordingly, one embodiment of the present inventioncomprises a method of treating sperm cells by adding at least one“organic stress reducing” agent (OSR) which may comprise an antioxidant,a vitamin or other organic molecule involved directly or indirectly inmodulating physiological stresses in the cell. The OSR would be added inthe concentration range of 0.01 mg/ml to 5 mg/ml to a sperm cell sampleto form a sperm cell composition. In certain embodiments, one or moreOSRs, each in the concentration range of 0.01 mg/ml to 5 mg/ml, can beadded to the sperm cell sample prior to cryopreservation (including, forexample, freezing and vitrification), after the sperm cell sample hasbeen thawed, or at both times. In other embodiments, the OSR can beadded at one or more of the various stages during the sperm cellprocessing procedure. The term “sperm cell sample” may comprise aprocessed semen sample or an unsorted, conventional semen sample.

Another specific embodiment of the invention comprises the sperm cellcomposition comprising a sperm cell sample and at least one OSR in theconcentration range of 0.01 mg/ml to 5 mg/ml. Another embodimentencompasses a “sperm cell composition” comprising a sperm cell sample,at least one OSR in the concentration range of 0.01 mg/ml to 5 mg/ml anda cryoprotectant. Most cryoprotectants can be used with the invention,including but not limited to egg yolk, propylene glycol, dimethylsulfoxide, sucrose, ethylene glycol and glycerol, or a combinationthereof. One embodiment encompasses a fresh, an unfrozen, a frozen, avitrified, or a thawed sperm cell composition comprising a sperm cellsample and at least one OSR in the concentration range of 0.01 mg/ml to5 mg/ml. Another embodiment encompasses a fresh, an unfrozen, a frozen,a vitrified, or a thawed sperm cell composition comprising a sperm cellsample, at least one antioxidant and/or one vitamin in the concentrationrange of 0.01 mg/ml to 5 mg/ml, and a stain.

Another broad object of the present invention is to improve themotility, viability (including longevity and ability to surviveenvironmental stress) and/or fertility of sperm cells, each contributingto the sperm cell's overall integrity, to improve the success of usingART, including techniques such as IVF, AI, ICSI (as well as othertechniques using enucleated cells), and MOET (as well as other embryotransfer techniques).

Such ART techniques involve different levels of gamete cell processingwhich in the case of sperm can entail, by example only and is notlimited to one or more of the following: artificially collecting a semensample from the male animal which may involve natural, electronic orother types of sexual stimulation; holding; transporting; buffering withdifferent pHs; chilling; warming; staining; diluting; concentrating;energetically exciting as with a laser; electronic charging; deflecting;ablating to kill unwanted cells usually with targeted lasers; sorting;collecting; shaking; oscillating; magnetically separating; oxygenatingas associated with microchip sorting procedures; labeling;precipitating; centrifuging; resuspending; mixing; dialyzing;cryostabilizing; freezing; vitrification; thawing; culturing;inseminating; microinjecting; microfluidic processing; microchipprocessing; jet and air processing; flow cytometry processing; andsimilar handling techniques. Whereas a single processing step may exertonly minimal stress on a sperm cell, others or a combination may addsignificant stress, often killing the cell. An extreme example is thesex-sorting process used to separate X- from Y-chromosome bearing cells;the sorting process combines a large number of independent highlystressful steps that severely compromise the overall integrity of thesorted sperm cell population.

Accordingly, one embodiment of the present invention resides broadly inthe use of a sperm cell composition, comprising a sperm cell sample andat least one antioxidant and/or at least one vitamin in theconcentration range of 0.01 mg/ml to 5 mg/ml, in ART. One specificembodiment of the invention comprises a method of increasing thepercentage of zygotes that develop into embryos in a given sample in agiven amount of time, as well as increasing the percentage of embryosthat are suitable for cryopreservation (i.e., the percentage of embryosthat are blastocysts, expanded and hatching blastocysts, or hatchedblastocysts), by mixing an egg with a sperm cell sample that has beentreated with at least one OSR in the concentration range of 0.01 mg/mlto 5 mg/ml. A further embodiment of the invention resides in a method ofmaking an embryo comprising mixing at least one egg with a sample ofsperm cells treated with at least one OSR in the concentration range of0.01 mg/ml to 5 mg/ml. The embryos produced by this method constitute afurther embodiment of the invention. Another embodiment includes amethod for inseminating an organism through an AI technique using asperm cell sample treated with at least one OSR in the concentration of0.01 mg/ml to 5 mg/ml. Another embodiment includes a method oftransferring an embryo into a receptive female (ET) where said embryo ismade using a sperm cell sample treated with at least one OSR in theconcentration of 0.01 mg/ml to 5 mg/ml. The progeny of the organism thatresults from the aforementioned AI method also constitutes an embodimentof the invention.

Most embodiments of the invention utilize concentrations of OSRsselected from the following ranges: 0.01 to 5.0 mg/ml; 0.01 to 0.25mg/ml; 0.01 to 0.5 mg/ml; 0.01 to 1 mg/ml; 0.01 to 2.5 mg/ml; 0.01 to 5mg/ml; 0.05 to 0.1 mg/ml; 0.05 to 1.0 mg/ml; 0.05 to 2.5 mg/ml; 0.1 to0.25 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 1 mg/ml; 0.1 to 2.5 mg/ml; 0.1 to 5mg/ml; 0.15 to 0.45 mg/ml; 0.15 to 0.5 mg/ml; 0.25 to 0.35 mg/ml; 0.25to 0.5 mg/ml; 0.25 to 1 mg/ml; 0.25 to 2.5 mg/ml; 0.25 to 5 mg/ml; 0.35to 0.5 mg/ml; 0.35 to 1 mg/ml; 0.35 to 2.5 mg/ml; 0.35 to 5 mg/ml; 0.5to 1 mg/ml; 0.5 to 2.5 mg/ml; 0.5 to 5 mg/ml; 1 to 2.5 mg/ml; 1 to 5mg/ml; about 0.05 mg/ml; about 0.1 mg/ml; about 0.15 mg/ml; about 0.25mg/ml; about 0.35 mg/ml; about 0.45 mg/ml; and about 0.5 mg/ml.

In some embodiments of the invention the sperm cell composition can beused immediately or processed within the first few minutes afteraddition of the OSR for whatever processing step is needed, whereby theholding period would be in the range 2 sec to 3 min. In otherembodiments, the sperm cell composition is held after the addition ofthe OSR(s) to allow the OSR(s) to incorporate into the cells andeffectuate protective effects on the cell population. Such holdingperiods can be short, as in the range of a 3-15 minutes, moderate as inthe range of 15 min to 1 hr; and longer processing periods ranging up toabout 8 hrs or overnight for extensive processing such as withsex-sorting techniques. Transportation hold periods associated withtransporting unfrozen sperm cell compositions can be much longer,extending up to a few days, which may for example occur if the sample iscollected, treated with the addition of one or more OSRs, transported orshipped to another location possibly by air, and further processed atthe second location as for sex-sorting at a designated facility. Inother instances, the sperm cell composition might need to be held for afew days while a recipient female is hormonally prepped for artificialinsemination, as might occur if a sample is mistakenly thawed and cannotbe refrozen. The addition of OSRs could theoretically prolong theseextended hold periods over what is currently accepted in the art, andcould provide sufficient protection to the sperm in the sperm cellcomposition so that they could remain viable and fertile for up to aweek or more.

In some embodiments of the invention, the OSR is added several timesduring a complex processing procedure to minimize cell stress throughoutthe procedure. In other embodiments, the OSR is added only at one ormore particular steps which are notably harsh on the cells to helpminimize stress and fatigue on the sperm cells. By way of example, thestaining process during sex sorting is often performed atnon-physiological pH and at elevated temperatures, both known to beharsh on the cells. Similarly, cryopreservation is also extremely harshon the cells and disrupts cell membranes, both internal and external.Following an intensive multi-step sorting procedure, sex-sorted spermcells which are already compromised are even more susceptible tocryogenic and freeze processing.

Various OSRs can be used in the context of the current invention,including but not limited to: catalase, superoxide dismutase (SOD), SODmimics, glutathione, glutathione reductase, glutathione peroxidase,pyruvate, mercaptoethanol, butylated hydroxytoluene (BHT), lipoic acid,flavins, quinines, vitamin K (and related vitamers), vitamin B12 (andrelated vitamers), with ‘vitamers’ defined as compounds having the samevitamin activity (such as cobalamin, cyanocobalamin, methylcobalamin,adenosylcobalamin, hydroxocobalamin, and pseudo-B12), vitamin E(including its vitamers, tocopherols (α, β, γ), tocotrienols, andα-tocopheryl), alpha-ketoglutarate (also known as α-KG, AKG oroxo-glutarate) and various biological forms of AKG (such as arginine,aspartate, lysine, and similar derivatives), other compounds thatregulate nitric oxide in the cell including malondialdehyde (MDA) andasymmetric dimethylarginine (ADMA) and biologically active derivativesthereof.

A further embodiment of the invention comprises a method of sorting asperm cell sample to form one or more subpopulations comprising thesteps of providing a sperm cell sample, sorting the sperm cell sample toform one or more subpopulations and adding at least one OSR to the spermcell sample during one or more of the aforementioned sorting steps, theconcentration of the OSR being in the range of 0.01 mg/ml to 5 mg/ml.

An additional embodiment of the invention encompasses media used inprocessing sperm cells that comprise at least one OSR at the appropriatestock concentration to be present at a final processing concentration inthe range of 0.01 mg/ml to 5 mg/ml in the sperm cell composition at thetime of processing. A stress reducing media can be used for differentprocesses including but not limited to sperm collection, artificialinsemination, sperm sorting, in vitro fertilization, embryo culture, aswell as sperm and embryo freezing. Media used in the sorting of spermcells typically comprise one or more buffers and/or extenders (i.e.,substances that preserve the viability and/or fertility of sperm cells).

Any buffer or buffer solution used in the processing of sperm can beused in the aforementioned media, including but not limited tophosphates, citrates, acetates, lactates, and combinations thereof, or asolution containing a salt, a carbohydrate, or a combination thereof canbe employed in some of the embodiments of the invention, such as, butnot limited to: Tris, TES, HEPES, TALP, TCA, PBS, citrate, milk andderivatives thereof, as discussed in detail in U.S. Pat. No. 7,208,265the contents of which is hereby incorporated by reference in itsentirety.

Any extender used in the processing of sperm can be used in theaforementioned media, including but not limited to energy sources,protein sources and antibiotics and may include one or more of thefollowing: mono- and disaccharides, such as fructose, glucose, mannose,sucrose, and lactose; protein sources, such as egg yolk, milk, BSA andderivatives thereof; and any one of the commonly known antimicrobial orantibiotic agents, such as gentamicin, lincomycin, spectinomycin, theirderivatives, or any combination thereof.

As used herein, the term “extender” may also include certain organicsubstances such as disaccharides, trisaccharides, and any combinationthereof, egg yolk, milk, albumin, lecithin, cholesterol, theirderivatives and any combination thereof. An extender may also include adetergent that may be an alkyl ionic detergent, such as sodium dodecylsulfate (SDS).

A further embodiment of the present invention provides a method ofimproving the motility, viability and/or fertility of a sperm cellsample that has already undergone a sorting process, including but notlimited to sex sorting, comprising the step of adding at least one OSRin the concentration range of 0.01 mg/ml to 5 mg/ml to a sorted spermcell sample to form a sperm cell composition.

Accordingly, the present invention resides broadly in the use of a spermcell composition, that in some embodiments comprise a sorted sperm cellsample and at least one OSR in the range of 0.01 mg/ml to 5 mg/ml, foruse in ART. A further embodiment encompasses a sperm cell compositioncomprising sorted sperm cells, at least one OSR in the concentrationrange of 0.01 mg/ml to 5 mg/ml and a catch media (i.e., media found inthe vessel that receives, or catches, the sorted sperm at the end of thesorting process). Another embodiment encompasses a sperm cellcomposition comprising a processed or sorted sperm cell sample, an OSRin the concentration range of 0.01 mg/ml to 5 mg/ml, and acryoprotectant. An additional embodiment of the invention encompasses afrozen or vitrified sperm cell composition comprising a processed orsorted sperm cell sample, and at least one OSR in the concentrationrange of 0.01 mg/ml to 5 mg/ml.

Another broad object of the present invention is to improve themotility, viability (including longevity and ability to surviveenvironmental stress) and fertility of processed and/or sorted spermcells for use in ART such as IVF, AI, ICSI (as well as other techniquesusing enucleated cells), and MOET (as well as other embryo transfertechniques).

Accordingly, some of the embodiments of the present inventionincorporate the use of sex sorted sperm cells that have had an OSR inthe concentration range of 0.01 mg/ml to 5 mg/ml added to them in ART.

Accordingly, other embodiments of the present invention incorporate theuse of a sperm cell composition, a sorted sperm cell sample, and atleast one OSR in the concentration range of 0.01 mg/ml to 5 mg/ml, inART.

One specific embodiment of the invention comprises a method ofincreasing the percentage of zygotes that develop into embryos in agiven sample in a given amount of time, as well as increasing thepercentage of embryos that are suitable for cryopreservation (i.e., thepercentage of embryos that are blastocysts, expanded and hatchingblastocysts, and hatched blastocysts), by mixing an egg with a sortedsperm cell sample that has been treated with at least one OSR in theconcentration range of 0.01 mg/ml to 5 mg/ml.

A further embodiment of the invention resides in a method of making anembryo comprising mixing at least one egg with at least one sperm celltreated with at least one OSR in the concentration range of 0.01 mg/mlto 5 mg/ml. The embryos produced by this method constitute a furtherembodiment of the invention.

Other embodiments of the invention also include, a method forinseminating an organism through an AI technique using a processed orsorted sperm cell sample treated with at least one OSR in theconcentration of 0.01 mg/ml to 5 mg/ml. The progeny of the organism thatresults from the aforementioned AI method also constitutes an embodimentof the invention. Furthermore, one embodiment of the inventionencompasses a method for recovering embryos that are produced from theaforementioned AI method.

Embodiments of the invention can include sperm cells, or spermatozoa,collected from numerous species of male mammals, and the inventionshould be understood not to be limited to the species of male mammalsdescribed by the specific examples within this application. Rather thespecific examples within this application are intended to beillustrative of the varied and numerous species of male mammals fromwhich semen can be collected and utilized in certain embodiments of theinvention. Embodiments of the invention, for example, may include thesperm cells of humans as well as animals having commercial value formeat or dairy production such as swine, ovine, bovine, equine, deer,elk, buffalo, or the like (naturally the mammals used for meat or dairyproduction may vary from culture to culture). It may also include thesperm cells of various domesticated mammalian species encompassed bycanines and felines, as well as sperm cells of primates, including butnot limited to chimpanzees, gorillas, or humans and the spermatozoa fromwhales, dolphins and other marine mammals. It may also includefrozen-thawed sperm cells from all the various mammals above-describedand further, including but not limited to, the sperm cells of deceaseddonors, from rare or exotic mammals, zoological specimens, or endangeredspecies.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, preferredembodiments of the present invention will now be described by way ofexample only with reference to the accompanying sheets of drawingswherein:

FIG. 1 is a schematic representation of part of a flow cytometerillustrating a method of sorting a sperm cell sample into one or moresubpopulations according to some embodiments of the present invention.

FIG. 2 illustrates a graphical representation of the motility andprogressive motility found in Table 1.

FIG. 3 is a graphical representation of the percent blastocysts andpercent hatching found in Table 4.

FIG. 4 is a graphical representation of the percent blastocysts andpercent hatching found in Table 5.

FIG. 5 is a graphical representation of the percent motile sperm foundin Table 1.

FIG. 6 is a graphical representation of the percent progressively motilesperm found in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, FIG. 1 illustrates in schematicform part of a flow cytometer used in a method to sort a sperm cellsample to form one or more subpopulations, the flow cytometer beinggenerally referenced 10. In this particular embodiment sex sorting istaking place so the subpopulations are X-chromosome bearing sperm cellsand Y-chromosome bearing sperm cells. FIG. 1 represents a singletechnique for sorting semen, but any known technique for sorting cellsknown in the art can be used with certain embodiments of the invention.Additional details of the basic sperm sorting apparatus and methodologyare described in U.S. Pat. Nos. 5,135,759 and 7,371,517, the contents ofwhich are hereby incorporated by reference in their entireties.

Once a sperm cell sample has been collected it can be extended as soonafter collection with an extender that includes one or more antioxidantsor vitamins. The sample is then typically held at a temperature of about5° C. for between about 12 hours to about 18 hours while it is beingshipped from the collection point to the flow cytometer 10 for thesorting process. This holding temperature can be in the range of between4° C. and 39° C. and is commonly in the range of 4° C. and 16° C.

Upon arrival at the flow cytometer, the sample is stained with a DNAselective dye and a quenching dye to form a stained sperm cell sampleand subsequently placed into a sperm cell source 11 of the flowcytometer 10. The flow cytometer 10 can be programmed by an operator togenerate two charged droplet streams, one containing X-chromosomebearing sperm cells, charged positively, 12, one containing Y-chromosomebearing sperm cells, charged negatively 13 while an unchargedundeflected stream of dead cells 14 simply goes to waste.

An operator may also choose to program the flow cytometer in such amanner, that both the X- and Y-chromosome bearing sperm are collectedusing a “high purity sort” (in other words only live X- and Y-chromosomebearing sperm are collected) or to program the flow cytometer to collectboth the X- and Y-chromosome bearing sperm using an “enriched sort” (inother words it will collect droplets containing live that were notpreviously sorted and excluding all initial dead again by the use ofBoolean Gate logic available with the computer that controls the flowcytometer). The Boolean Gate logic can also be used to collect only oneof either the X- or Y-chromosome bearing sperm.

Initially, a stream of sperm cells under pressure, is deposited into thenozzle 15 from the sperm cell source 11 in a manner such that they areable to be coaxially surrounded by a sheath fluid supplied to the nozzle15 under pressure from a sheath fluid source 16. An oscillator 17 whichmay be present can be very precisely controlled via an oscillatorcontrol mechanism 18, creating pressure waves within the nozzle 15 whichare transmitted to the coaxially surrounded sperm cell stream as itleaves the nozzle orifice 19. As a result, the exiting coaxiallysurrounded sperm cell stream 20 could eventually and regularly formdroplets 21.

The charging of the respective droplet streams is made possible by thecell sensing system 22 which includes a laser 23 which illuminates thenozzle exiting stream 20, and the light emission of the fluorescingstream is detected by a sensor 24. The information received by thesensor 24 is fed to a sorter discrimination system 25 which very rapidlymakes the decision as to whether to charge a forming droplet and if sowhich charge to provide the forming drop and then charges the droplet 21accordingly.

A characteristic of X-chromosome bearing sperm is that they tend toabsorb more fluorochrome dye than Y-chromosome bearing sperm and assuch, the amount of light emitted by the laser excited absorbed dye inthe X-chromosome bearing sperm differs from that of the Y-chromo-somebearing sperm and this difference in characteristic tells the sorterdiscrimination system 25 which charge to apply to droplets containingonly X- or only Y-chromosome bearing sperm cells. Dead cells (or thoseabout to die) have absorbed the quenching dye and the sorterdiscrimination system 25 does not charge droplets containing such cells.

The charged or uncharged droplet streams then pass between a pair ofelectrostatically charged plates 26, which cause them to be deflectedeither one way or the other or not at all depending on their charge intorespective collection vessels 28 and 29 to form respectively a genderenriched population of X-chromosome bearing and a gender enrichedY-chromosome bearing sperm cells having a DNA selective dye associatedwith their DNA. The uncharged non-deflected stream containing asub-population of dead cells (or those that are about to die) go to thewaste container 30.

The collected sex sorted sperm cells may then be frozen and stored orfrozen and sent on for further processing (or simply used for furtherprocessing immediately), further processing meaning for example, thepurposes of research or for use in ART such as IVF, AI, ICSI (as well asother techniques using enucleated cells), and MOET (as well as otherembryo transfer techniques).

In alternative embodiments not illustrated, the catch media contained inthe otherwise empty collection vessels may also contain OSR in theconcentration range of 0.01 mg/ml to 5 mg/ml. The OSR may be addedduring this stage of the sorting process (be it sex sorting or otherform of sorting) and/or in addition to another method step in thesorting (be it sex sorting or other form of sorting) process.

Furthermore, in the alternative embodiments, the OSR administered in theconcentration range of 0.01 mg/ml to 5 mg/ml to the sperm cell sample orcomposition can be added to the DNA selective dye and/or the quenchingdye solutions. Some embodiments include use of one or more OSRs aspre-mixed components of the prepared buffers, extenders, stains, catchfluids, and/or cryo-extenders used in the sex sorting procedure.Accordingly, the OSR may be added to the sperm cell sample at one ormore steps during sex-sorting, including when the sperm cell sample isbeing first handled following collection, and/or stained with a DNAselective dye and/or the quenching dye, and/or at the time of collectionfrom the flow cytometer, and/or later when preparing the sample forcryopreservation by adding the OSR or OSR cocktail to the cryoextender.

Likewise, some sorting embodiments include sorting of frozen-thawedconventional semen whereby the OSR can be added to the thawed semensample shortly after thawing and then reverse sorted to producesex-sorted sperm cell subpopulations which include the addition of anOSR at one or more steps during the extended processing procedure ofgender selection.

In some cases, when the sorting of sperm cells is not going to involvesex sorting, a quenching dye without the need for a DNA staining dye maybe required, in which case the OSR will only be present in the quenchingdye to form the stained sample. In this way, depending on the embodimentchosen, the OSR may again only be added during this stage of the sortingprocess (be it sex sorting or other form of sorting) or in addition toat least one other method step in the sorting (be it sex sorting orother form of sorting) process.

Again, in the alternative embodiments, the collected sex sorted spermcells (or in alternative embodiments the sorted, i.e. non-sex sortedsperm cell samples) once frozen will, prior to such further processing,require to be thawed. Either before freezing or upon thawing, theantioxidant, again in the concentration range of 0.01 mg/ml to 5 mg/ml,may be added to the sample before freezing and/or to the thawed sample.In this way, depending on the embodiment chosen, the OSR may again onlybe added during this stage of the sorting process (be it sex sorting orother form of sorting) or in addition to at least one other method stepin the sorting (be it sex sorting or other form of sorting) process.

In yet further alternative embodiments the time period allowed to elapseafter the addition of the OSR, can vary and may be in the range of about5 seconds to about 72 hours (excluding freezing time and time spent inthe freezer or in the cryopreserved state), the lower end of the scaleproviding for almost immediate sorting of the sperm sample while theupper end of the scale would cover the typical maximum time frameassociated with moving a sperm sample from its collection point to itssorting point. This is usually a flight and/or road travel time. Moreparticularly, the time periods may be about 5 seconds to about 3 hours;about 3 hours to about 6 hours; about 6 hours to about 12 hours; about12 hours to about 18 hours; about 18 hours to about 24 hours; about 24hours to about 36 hours; about 36 hours to about 48 hours; about 48hours to about 60 hours; and about 60 hours to about 72 hours; and.

Another alternative embodiment may include the use of an extender withina pH range of 5.5 to 7.8, and frequently at about 6.5; about 6.6; about6.7; about 6.8; about 6.9; about 7.0; about 7.1; about 7.2; about 7.3;about 7.4; or about 7.5.

As described in the illustrated embodiment, different steps of themethod are carried out at different temperatures. In alternativeembodiments, at least one of the method steps is carried out within atemperature range selected from the group consisting of about 5° C. toabout 15° C.; about 15° C. to about 20° C.; about 20° C. to about 25°C.; about 25° C. to about 30° C.; about 30° C. to about 35° C.; about35° C. to about 40° C. and about 40° C. to about 45° C. This allows fordifferent steps in the sorting method to be performed within differenttemperature ranges.

In another aspect of the present invention there is provided a method oftreating the motility of sperm cells in a sperm cell sample. In thisembodiment, the sperm cell sample, which may be a gender enrichedpopulation of X-chromosome bearing or Y-chromosome bearing sperm cellshaving a DNA selective or DNA binding dye associated with their DNA, asample sorted into one or more subpopulations or a conventionalnon-sorted sample, the method of treating the motility of the spermcells in the sperm cell sample comprises the step of adding an OSR inthe concentration range of 0.01 mg/ml to 5 mg/ml to the sperm cellsample to form a sperm cell composition (and in this embodiment at aconcentration of 0.5 mg/ml). The OSR which is added forms part of anextender which is in a pH range of 6.5 to 7.5 and in particularembodiments, the pH is selected from the group consisting of about 6.5;about 6.6; about 6.7; about 6.8; about 6.9; about 7.0; about 7.1; about7.2; about 7.3; about 7.4; and about 7.5.

After the addition of the antioxidant, a time period being in the rangeof about 5 seconds to about 72 hours is allowed to elapse before thesample undergoes further processing in the form of for example, researchor for use in assisted reproductive technologies such as IVF, AI, ICSI(as well as other techniques using enucleated cells), and MOET (as wellas other embryo transfer techniques). The time period which is allowedto elapse may be selected from the range consisting of: about 5 secondsto about 3 hours; about 3 hours to about 6 hours; about 6 hours to about12 hours; about 12 hours to about 18 hours; about 18 hours to about 24hours; about 24 hours to about 36 hours; about 36 hours to about 48hours; about 48 hours to about 60 hours; and about 60 hours to about 72hours. The techniques for fertilizing an egg involve the added step ofmixing at least one egg with the sperm cell sample. Any conventionaltechnique such as those listed above can be used with the invention,including any conventional IVF or AI technique. Typical IVF techniquesare disclosed in WO/0243486, for example, which is incorporated byreference herein in its entirety. Typical AI techniques are disclosed inU.S. Pat. No. 6,149,867, for example, which is incorporated by referenceherein in its entirety.

In alternative embodiments, the sperm cell sample may have been a frozensample that has been allowed to thaw. The method may further comprisethe step of staining the sperm cell sample or the sperm cell compositionwith a DNA selective dye if the sample is not a sex sorted sample.

The method may also comprise the step of freezing the sperm cellcomposition to form a frozen sperm cell composition that may be allowedto thaw. At least one of the method steps in this second aspect of thepresent invention is carried out within a temperature range selectedfrom the group consisting of about 0° C. to about 5° C.; about 5° C. toabout 15° C.; about 15° C. to about 20° C.; about 20° C. to about 25°C.; about 25° C. to about 30° C.; about 30° C. to about 35° C.; about35° C. to about 40° C. and about 40° C. to about 45° C. Thus, eachmethod step may be carried out at a different or similar temperaturerange.

Suggested Methods

By way of example, the following oocyte maturation procedure, IVFprocedure, in vitro culture procedure and co-culture procedure may beused with the invention. One skilled in the art will know thatvariations on these methods exist and that these methods should not beconstrued to limit the functionality of the current invention. Thesemethods are illustrative only.

1. Oocyte Collection.

Collect slaughterhouse oocytes, wash 1× with about 3 mL Hepes washingmedia and with 1× with TCM-199 (Invitrogen, Carlsbad, Calif.)+10% FetalBovine Serum (FBS). Culture in maturation media for 22 hrs in a CO₂incubator at 38.5° C. In one embodiment, the maturation media containsTCM-199, FBS, pyruvate, chorionic gonadotropin (e.g., Chorulon(Intervet, Summit N.J.)), follicle stimulating hormone (FSH) (e.g.,Folltropin (Bioniche, Belleville, Canada)), estradiol, and at least oneantibiotic. In a further embodiment, Amikacin (Sigma-Aldrich, St. Louis,Mo.) can be used as the antibiotic. In another embodiment, thematuration media may also comprise luteinizing hormone.

In one embodiment, the maturation media may comprise 5-20 ml of TCM-199Earl's; 0.5-2 ml of FBS (Thermo Fisher Scientific, Waltham, Mass.);10-30 μl of pyruvate (prepared by adding 0.05-0.20 g of sodium pyruvate(Sigma-Aldrich, St. Louis, Mo.) to 5-20 ml of saline solution); 50-200μl of chorionic gonadotropin (prepared by adding 5-20 UI of Chorulon(Intervet, Summit N.J.) to 5-20 ml of TCM-199 Earl's); 5-20 μl of FSH(prepared by adding 0.001-0.01 g of Folltropin (Bioniche, Belleville,Canada) to 5-20 ml of TCM-199 Earl's); 5-20 μl of estradiol (prepared byadding 0.001-0.05 g of estradiol (Sigma-Aldrich, St. Louis, Mo.) to 5-20ml of Etanol (Sigma-Aldrich, St. Louis, Mo.)); and 10-30 μl Amikacin(prepared by adding 0.1-1 g Amikacin sulfate salt (Sigma-Aldrich) to20-40 ml of saline solution). In alternative embodiments, the maturationmedia may comprise the aforementioned components using different volumesbut in the same proportion to each other, e.g., in one embodiment, thematuration media may comprise 10-40 ml of TCM-199; 1-4 ml of FBS; 20-60μl of sodium pyruvate, etc. In a further embodiment, the maturationmedia comprises the above preparations of TCM-199 Earl's, FBS, pyruvate,chorionic gonadotropin, FSH, estradiol and an antibiotic in theapproximate ratio of 9:1:0.02:0.1:0.01:0.01:0.02 by volume,respectively.

2. In Vitro Fertilization.

Trim away cumulus cells from matured oocytes. Transfer them to afertilization dish and return to the CO₂ incubator. Thaw frozen semenstraws using standard procedures, centrifuge in 800 μL of Pure Spermgradient (Nidacon, Molndal, Sweden), or a percoll or similar gradient at2500 RPM for 10 minutes to remove egg components, glycerol and otherdebris. Remove supernatant, leaving a loose pellet of live sperm.Combine pellets using a small amount of fertilization media andrepellent at 1500 RPM for 3 minutes. Carefully remove supernatant. Thengently mix the pellet. After determining the desired insemination dose,inseminate the oocytes by adding sperm to the pellet, then culture in adish and return to the CO₂ incubator for about 18-22 hours.

3. In Vitro Culture.

Remove presumptive zygotes from the fertilization dish and transfer intoa sterile 1.5 mL eppendorf tube. Allow zygotes to form a loose pelletand remove excess media to form a 1:1 ratio of pellet and solution.Rinse the eppendorf tube with TCM-199, place contents into a dish andwash with BSA media. Then culture presumptive zygotes (discarddisfigured oocytes, as well as oocytes with yellow colored cytoplasm orvacuolated cytoplasm) in a dual gas incubator (5% CO₂, 5% O₂) at 38.5°C. for about 48 hours.

4. Co-Culture.

Transfer cleaved zygotes to co-culture dishes comprising the cumuluscells from the mature oocytes and FBS media topped with mineral oil, andincubate in a CO₂ incubator at 38.5° C. until needed.

5. Sperm Motility Evaluations by CASA.

A comparison of viewing chambers and slides can be done in a variety ofIVOS instruments, which for example only can be a Hamilton-Thorne IVOS(Hamilton-Thorne, Beverly, Mass.). Instrument settings: image capture;frames per second=60; number of frames=30; cell detection; minimumcontrast=50; minimum cell size=5; defaults, cell size=5; cellintensity=50; progressive cells, path velocity=50 um/s; straightness≧70%; slow cells (um/s); average path velocity (VAP, <30 um/s),straight-line velocity (VSL, <15 um/s). The CASA motility variablesmeasured can be a percentage of total motile sperm (motile), percentageof progressively motile sperm (progressive), VAO, VSL, curvilinearvelocity (VCL, um/s), average lateral head displacement (ALK, um) andthe number of times the sperm head crosses the mean path/s (BCF, Hz),straight-line sperm motility (STR, %), and linear sperm motility (LIN,%). See for instance, Lenz, R W, et al., J Anim Sci (2011) 89:383-388,incorporated by reference herein in its entirety.

A further aspect of the present invention entails the use of a spermcell sample treated with an OSR in AI. AI in the present inventionincludes a method whereby a fresh or frozen thawed sperm cell sample isused to inseminate by way of passage of the semen or sperm sample intothe female reproductive tract, with or without an accessorizing toolsuch as an AI gun, catheter or pipette.

Frozen semen samples may be contained in semen straws, which are thawedbefore the AI procedure using standard methods. In certain embodimentsof the invention, the semen straws contain about 0.25-0.5 ml of fluidand are often sufficient for a single insemination.

To increase the number of offspring that a female can produce, embryotransfer techniques (such as MOET) have been developed and are wellknown to those skilled in the art. Conventional embryo transfertechniques include injection of females with suitable hormones thatcause them to produce multiple eggs (oocytes) in a single estrous cycle.This process is often referred to as superovulation. Each female is thenartificially inseminated with a sperm cell sample from a male that iseither fresh or has been cryopreserved.

In another aspect of the invention, zygotes and/or embryos fromartificially inseminated females can be recovered and then culturedand/or cryopreserved/vitrified.

Example 1

One set of semen from each bull was used as a control while to theremaining sets of semen samples were added vitamin B12, as theantioxidant, at respective concentrations of 0.5 mg/ml and 0.25 mg/ml.For each sample, the same concentration of vitamin B12 was added (i)during the staining process, and (ii) in the catch fluid of thecollection vessel. Control samples did not contain vitamin B12. Spermsamples were sorted in “High Purity” mode, and the collected sex-sortedsperm were extended with a cryoextender, which in some cases againcontained the same concentration of vitamin B12 and the samples werefrozen. Three hours after thawing (which is a standard time frame toconduct quality control assessments on sorted frozen thawed spermsamples) the thawed samples were put through CASA, a machine thatprovides various data on sperm including motility and progressivemotility information. The results are shown below in Table 1 and themotility and progressive motility are graphically represented in FIG. 2.

In the tables below: VAP (average path velocity (μm/s)); VSL(straight-line velocity (μm/s)); VCL (curvilinear velocity (μm/s)); ALH(average lateral head displacement (μm)); BCF (the number of times thesperm head crosses the mean path/s in Hz); STR (percent straight-linesperm motility); LIN (percent linear sperm motility); PIA (percentintact acrosomes); motile (percent motile sperm); and progressive(percent progressively motile sperm).

The two step addition results represent treatments with vitamin B12present at the same indicated concentration in the catch fluid of thecollecting vessel and in the cryoprotectant extender prior to freezingthe sample only (2 step-freeze; −++); the three step addition resultsindicate that the same concentration of vitamin B12 was added during thestaining step, the collecting step (in the catch fluid of the collectingvessel) and in the cryoprotectant extender prior to freezing the sample(3 step). There is no correlation with regard to randomly assigned namessuch as “Bull A.” Bull A from Example 5 is not the same bull as Bull Afrom Example 7.

TABLE 1 Motility (2 step and 3 step) (3 hrs Post-Thaw) Total Motile ProgMot VAP VSL VCL ALH BCF STR LIN PIA cells (%) (%) (μm/s) (μm/s) (μm/s)(μm) (Hz) (%) (%) (%) Bull 1 Control 562 59.5 33.5 54 47 95.5 4 21.587.5 51 73.5 0.5 mg/ml, 2 step F 925 63.5 26 56 45 94 4 23 86.5 49 740.25 mg/ml, 3 step 815 70.5 40.5 55 48 92 4 23.5 87.5 53 83 Bull 2Control 529 54.5 41.5 61 53.5 107.5 4.5 23 87.5 51.5 64 0.5 mg/ml, 2step F 269 70.5 47 77 66 108 4 21.5 85.5 61 84 0.25 mg/ml, 3 step 693 6753.5 68 55 111.5 4.5 24 87 51 79.5 Bull 3 Control 599 47.5 18 49.5 42.584.5 4 20.5 86.5 51.5 52.5 0.5 mg/ml, 2 step F 800 74.5 53.5 56 51 98 422.5 84.5 52.5 87 0.25 mg/ml, 3 step 656 70 48.5 59 50 101 4 24 85.5 5180 Bull 4 Control 602 65.5 33.5 53.5 45.5 89.5 4 23.5 86 52.5 72.5 0.5mg/ml, 2 step F 874 81.5 50 60 53 109 5 26 83.5 49 88 0.25 mg/ml, 3 step1059 81 25.5 43 51 88.5 5 23 82.5 47 86 Bull 5 Control 638 49.5 13 45.538.5 86.5 5 18 84 45.5 63.5 0.5 mg/ml, 2 step F 842 51 33 50 47 101 520.5 84 47 79 0.25 mg/ml, 3 step 787 70 40.5 61 48 106.5 5 21.5 84.5 4780

Example 2

In another series of experiments, the CASA results regarding motilityand progressive motility after 4.5 hours after thawing sex-sortedsamples against a control are shown below in Table 2. The (2step-freeze) results indicate that vitamin B12 at the same concentrationwas present in the catch fluid of the collecting vessel and in thecryoprotectant extender prior to freezing the sample; the (3 step)results indicate that the same concentration of vitamin B12 was addedduring the staining step, the collecting step (in the catch fluid of thecollecting vessel) and in the cryoprotectant extender prior to freezingthe sample.

With respect to “Bull A,” the two sets of results shown were obtained ontwo different days using thawed samples from the same initially sortedbatch of sperm. In both cases the concentration of the vitamin B12 was 1mg/ml. For all other bulls in the table below unless otherwiseindicated, the concentration of vitamin B12 added was also 1 mg/ml.

TABLE 2 Motility (4.5 hrs Post-Thaw) Total cells Motile Progressive BullA control 322 38 0 1 mg/ml - 2 step-F 250 41 8 1 mg/ml - 3 step 287 6437 Bull A (day 2) control 328 46 4 1 mg/ml - 2 step-F 433 71 31 1mg/ml - 3 step 487 64 11 Bull B control 298 33 1 1 mg/ml - 2 step-F 31622 1 1 mg/ml - 3 step 237 59 19 Bull C control 403 52 1 1 mg/ml - 2step-F 432 76 43 1 mg/ml - 3 step 613 62 2 Bull D control 644 37 3 1mg/ml - 3 step 400 59 9 Bull E control 653 41 1 1 mg/ml - 3 step 648 521 Bull F control 638 39 7 0.5 mg/ml - 3 step 761 51 16 0.25 mg/ml - 3step 730 60 23 Bull G control 588 41 13 0.5 mg/ml - 3 step 162 65 570.25 mg/ml - 3 step 1003 56 32

Example 3

In an additional experiment, motility and progressive motility werechecked 3.75 hours after thawing a sex sorted semen sample treated withthe antioxidant. The OSR was not added to the control sample. The semensample was derived from a single bull. 0.25 mg/ml concentration ofvitamin B12 was added to the test sample during the staining step, thecollecting step (in the catch fluid of the collecting vessel) and in thecryoprotectant extender prior to freezing the sample. The results areshown in Table 3 below.

TABLE 3 Motility (3.75 hrs Post-Thaw) Total Motile Prog Mot VAP VSL VCLALH BCF STR LIN PIA Bull 1 cells (%) (%) (μm/s) (μm/s) (μm/s) (μm) (Hz)(%) (%) (%) Control 452 7 0 37 28 71 3 16 74 40 41 0.25 mg/ml, 3 step536 39 5 40 34 76 5 17 85 45 70

Example 4

An experiment was designed to test the effect of OSR treated sperm cellson the development of eggs fertilized with those sperm cells. In thisexperiment, bull semen from two bulls was sex sorted with flowcytometry, with vitamin B12 present at equal concentrations in the mediaused to stain the sperm cells, in the catch fluid of the collectingvessel and in the cryoprotectant extender prior to freezing the sexsorted sample (3 step). For each bull, three different concentrations ofvitamin B12 were tested: 0.05 mg/ml, 0.15 mg/ml and 0.25 mg/ml. Thecontrol samples were not treated with vitamin B12.

Slaughterhouse oocytes were collected and washed 1× with about 3 mLHepes washing media and with 1× with TCM-199+10% FBS. The oocytes werethen cultured in maturation media for 22 hrs in a CO₂ incubator at 38.5°C. Cumulus cells were trimmed away from matured oocytes, transferred toa fertilization dish, and returned to the CO₂ incubator.

Frozen semen straws were thawed using standard procedures andcentrifuged in 800 μL of Pure Sperm gradient at 2500 RPM for 10 minutesin order to remove egg, glycerol and other debris. The supernatant wasremoved, leaving a loose pellet of live sperm. Pellets were combinedusing a small amount of fertilization media and repelleted at 1500 RPMfor 3 minutes. The supernatant was then carefully removed and the pelletgently mixed. After determining the desired insemination dose, thematured oocytes were then inseminated by adding sperm to the pellet,cultured in a dish and returned to the CO₂ incubator for about 18-22hours.

Presumptive zygotes were removed from the fertilization dish andtransferred into a sterile 1.5 ml eppendorf tube. The zygotes wereallowed to form a loose pellet and excess media was removed to form a1:1 ratio of pellet and solution. The eppendorf tube was vortexed for 90seconds and then rinsed with TCM-199. The contents were placed into adish and then washed with BSA media. The presumptive zygotes were thencultured in a dual gas incubator (5% CO₂, 5% O₂) at 38.5° C. for about48 hours. Cleaved zygotes were then transferred to co-culture dishescomprising the cumulus cells from the mature oocytes and FBS mediatopped with mineral oil and incubated in a CO₂ incubator at 38.5° C.

Embryos were observed 7 days after IVF to check: Zyg (the number ofzygotes put into culture); 4-2C (the number of zygotes that underwentthe 2 cell to 4 cell transition 48 hours after IVF); 8C (the number ofzygotes with 8 cells 48 hours after IVF); 8C % (the percentage ofzygotes having 8 cells 48 hours after IVF); % C1v (percent cleaved 48hours after IVF); C1 (number of expanded and hatching and hatchedblastocysts 7 days after IVF); C1− (number of blastocysts 7 days afterIVF); and C2 (number of early blastocysts and compact morulas 7 daysafter IVF), Total Embs (total number of blastocysts=C1+C1−+C2); Blast %(percent of cultured zygotes resulting in blastocyst formation); Hatch #(the number of embryos shedding the zona palucida in preparation forimplantation observed at 8.5 days after IVF); and Hatch % (percentage ofembryos that shed the zona). The results are provided in Table 4 below.

TABLE 4 IVF - Embryo/Fertilization (3 step) Total Blast Hatch Hatch Zyg4-2C 8C 8C % % Clv C1 C1− C2 Embs % # % Bull A Control 96 36 8 8.3 45.81 0 5 6 6.3 2 33.3 Control 68 19 7 10.3 38.2 0 0 2 2 2.9 0 0.0 Control91 31 19 20.9 54.9 2 3 10 15 16.5 5 33.3 Total/Avg 255 86 34 (13.3)(47.1) 3 3 17 23 (9.0) 7 (30.4) 0.05 mg/ml 87 20 3 3.4 26.4 0 0 1 1 1.10 0.0 0.05 mg/ml 95 26 9 9.5 36.8 1 0 1 2 2.1 1 50.0 0.05 mg/ml 94 20 1111.7 33.0 0 3 1 4 4.3 1 25.0 Total/Avg 276 66 23 (8.3) (32.2) 1 3 3 7(2.5) 2 (28.6) 0.15 mg/ml 89 25 4 4.5 32.6 0 1 4 5 5.6 0 0.0 0.15 mg/ml90 29 9 10.0 42.2 1 0 4 5 5.6 3 60.0 0.15 mg/ml 91 24 6 6.6 33.0 2 1 4 77.7 3 42.9 Total/Avg 270 78 19 (7.0) (35.9) 3 2 12 17 (6.3) 6 (35.3)0.25 mg/ml 91 33 6 6.6 42.9 0 2 3 5 5.5 2 40.0 0.25 mg/ml 93 38 20 21.562.4 4 2 10 16 17.2 10 62.5 0.25 mg/ml 91 26 16 17.6 46.2 2 3 10 15 16.56 40.0 Total/Avg 275 97 42 (15.3) (50.5) 6 7 23 36 (13.1) 18 (50.0) BullB Control 91 15 35 38.5 54.9 3 3 8 14 15.4 9 64.3 Control 90 25 29 32.260.0 4 5 9 18 20.0 10 55.6 Control 92 20 33 35.9 57.6 7 6 12 25 27.2 1560.0 Total/Avg 273 60 97 (35.5) (57.5) 14 14 29 57 (20.9) 34 (59.6) 0.05mg/ml 90 18 40 44.4 64.4 2 4 13 19 21.1 9 47.4 0.05 mg/ml 92 23 27 29.354.3 4 2 9 15 16.3 7 46.7 0.05 mg/ml 93 22 27 29.0 52.7 5 3 13 21 22.6 838.1 Total/Avg 275 63 94 (34.2) (57.1) 11 9 35 55 (20.0) 24 (43.6) 0.15mg/ml 96 21 38 39.6 61.5 6 7 10 23 24.0 17 73.9 0.15 mg/ml 93 32 14 15.149.5 2 3 6 11 11.8 6 54.5 0.15 mg/ml 100 21 29 29.0 50.0 4 4 14 22 22.09 40.9 Total/Avg 289 74 81 (28.0) (53.6) 12 14 30 56 (19.4) 32 (57.1)0.25 mg/ml 92 24 34 37.0 63.0 5 3 11 19 20.7 14 73.7 0.25 mg/ml 96 31 2324.0 56.3 1 3 3 7 7.3 5 71.4 Total/Avg 188 55 57 (30.3) (59.6) 6 6 14 26(13.8) 19 (73.1)

Example 5

A similar experiment as done in Example 4 was done to test the effect ofOSR treated sperm cells on the development of eggs fertilized using ahigher concentration of the antioxidant, comparing it to one of theearlier used concentrations. Semen samples from two bulls of differentbreeds (one Holstein; one Jersey) were sex sorted using flow cytometry,again using vitamin B12 as the antioxidant, present at equalconcentrations during staining, in the catch fluid of the collectingvessel and in the cryoprotectant extender prior to freezing the sexsorted sample (3 step). For each bull, the two concentrations of vitaminB12 tested were: 0.5 mg/ml and 0.25 mg/ml. The control samples were nottreated with vitamin B12. All experimental steps were done the same asin Example 4. The results are shown in Table 5 below.

TABLE 5 IVF - Embryo Fertilization (3 step) Total Blast Hatch Hatch Zyg4-2C 8C 8C % % Clv C1 C1− C2 Embs % # % Bull A (Holstein) Control 91 2535 38.5 65.9 3 2 4 9 9.9 3 33.3 Control 93 40 19 20.4 63.4 1 2 3 3.2 00.0 Control 98 25 25 25.5 51.0 3 6 5 14 14.3 6 42.9 Total/Avg 282 90 7928.0 (60.1) 6 9 11 26 (9.2) 9 (34.6) 0.25 mg/ml 97 25 39 40.2 66.0 6 9 520 20.6 8 40.0 0.25 mg/ml 97 34 30 30.9 66.0 5 6 5 16 16.5 4 25.0 0.25mg/ml 97 23 33 34.0 57.7 6 7 6 19 19.6 9 47.4 Total/Avg 291 82 102 35.1(63.2) 17 22 16 55 (18.9) 21 (38.2)  0.5 mg/ml 94 25 34 36.2 62.8 0 3 36 6.4 1 16.7  0.5 mg/ml 95 29 30 31.6 62.1 1 7 7 15 15.8 4 26.7  0.5mg/ml 93 18 35 37.6 57.0 5 7 8 20 21.5 11 55.0 Total/Avg 282 72 99 35.1(60.6) 6 17 18 41 (14.5) 16 (39.0) Bull B (Jersey) Control 97 39 39 40.280.4 5 5 3 13 13.4 7 53.8 Control 100 22 12 12.0 34.0 4 5 9 9.0 2 22.2Control 100 22 29 29.0 51.0 2 5 1 8 8.0 3 37.5 Total/Avg 297 83 80 26.9(55.1) 11 15 4 30 (10.1) 12 (40.0) 0.25 mg/ml 94 25 45 47.9 74.5 5 9 418 19.1 12 66.7 0.25 mg/ml 99 36 20 20.2 56.6 7 6 6 19 19.2 6 31.6 0.25mg/ml 99 17 46 46.5 63.6 5 7 8 20 20.2 9 45.0 Total/Avg 292 78 111 38.0(64.8) 17 22 18 57 (19.5) 27 (47.4)  0.5 mg/ml 100 40 34 34.0 74.0 4 6 111 11.0 5 45.5  0.5 mg/ml 95 31 24 25.3 57.9 5 11 7 23 24.2 6 26.1  0.5mg/ml 99 15 46 46.5 61.6 8 5 5 18 18.2 9 50.0 Total/Avg 294 86 104 35.4(64.5) 17 22 13 52 (17.7) 20 (38.5)

Example 6

Another experiment similar to Examples 4 and 5 was performed to test thereproducible effect of OSR treated sperm cells regarding a single bullby monitoring the development of embryos. Bull A (Holstein) was sampledthree different times; Bull B was sampled five different times; Bull Conly one time; all semen samples were subjected to standard sex sortingprocedures using flow cytometry with vitamin B12 as the OSR at 0.25mg/ml during the steps of staining, collection in the catch fluid andprior to cryopreservation (3 step). The control samples were not treatedwith vitamin B12. All experimental steps were done the same as inExample 4. The results are provided in Table 6, below.

TABLE 6 IVF (3 step) - Reproducibility Total Blast Freeze Hatch Zyg 4-2C8C 8C % % Clv C1 C1− C2 Embs % % % Bull A (Holstein) Trial 1 Control 50785 119 23.5 40.2 34 19 31 84 16.6 10.5 0.25 mg/ml 434 92 100 23.0 44.227 14 44 85 (19.6) 9.4 Trial 2 Control 993 238 287 28.9 52.9 54 26 86166 16.7 8.1 0.25 mg/ml 884 204 222 25.1 48.2 81 36 128 245 (27.7) 13.2Trial 3 Control 907 209 215 23.7 46.7 70 27 90 187 20.6 10.7 0.25 mg/ml1053 230 285 27.1 48.9 83 39 129 251 (23.8) 11.6 Average - Control(18.2) (9.6) Average - 0.25 mg/ml (24.5) (11.8) Bull B (Holstein) Trial1 Control 507 85 119 23.5 40.2 34 19 31 84 16.6 10.5 0.25 mg/ml 434 92100 23.0 44.2 27 14 44 85 (19.6) 9.4 Trial 2 Control 993 238 287 28.952.9 54 26 86 166 16.7 8.1 0.25 mg/ml 884 204 222 25.1 48.2 81 36 128245 (27.7) 13.2 Trial 3 Control 907 209 215 23.7 46.7 70 27 90 187 20.610.7 0.25 mg/ml 1053 230 285 27.1 48.9 83 39 129 251 (23.8) 11.6 Trial 4Control 550 122 177 32.2 54.4 12 19 56 87 15.8 5.6 0.25 mg/ml 595 124183 30.8 51.6 12 8 52 72 (12.1) 3.4 Trial 5 Control 596 107 159 26.744.6 31 21 83 135 22.7 8.7 0.25 mg/ml 636 137 185 29.1 50.6 27 24 79 130(20.4) 8.0 Average - Control (18.5) (8.8) Average - 0.25 mg/ml (21.7)(9.7) Bull C Trial 1 Control 454 113 265 58.4 83.3 21 17 49 87 (19.2)(8.4) 0.25 mg/ml 509 104 335 65.8 86.2 31 27 62 120 (23.6) (11.4)

Example 7

A larger scale experiment was done to further test the reproducibilitysimilar to what was done in Examples 6 and 7, but testing five bullseach three separate times. Semen samples from five bulls were sampledand sex sorted using flow cytometry in accordance with the earlierprocedures and treated with 0.25 mg/ml vitamin B12 as the OSR (3 step).The control samples were not treated with vitamin B12.

TABLE 7 Averaged Effect (Holstein and Jersey Mix) Total Blast HatchHatch Zyg 4-2C 8C 8C % % Clv C1 C1− C2 Embs % # % Control-A1 993 238 28728.9 52.9 54 26 86 166 16.7 Control-A2 507 85 119 23.5 40.2 34 19 31 8416.6 Control-A3 907 209 215 23.7 46.7 70 27 90 187 20.6 Control-B1 96 368 8.3 45.8 1 0 5 6 6.3 2 33.3 Control-B2 68 19 7 10.3 38.2 0 0 2 2 2.9 00.0 Control-B3 91 31 19 20.9 54.9 2 3 10 15 16.5 5 33.3 Control-C1 91 1535 38.5 54.9 3 3 8 14 15.4 9 64.3 Control-C2 90 25 29 32.2 60.0 4 5 9 1820.0 10 55.6 Control-C3 92 20 33 35.9 57.6 7 6 12 25 27.2 15 60.0Control-D1 97 39 39 40.2 80.4 5 5 3 13 13.4 7 53.8 Control-D2 100 22 1212.0 34.0 4 5 9 9.0 2 22.2 Control-D3 100 22 29 29.0 51.0 2 5 1 8 8.0 337.5 Control-E1 91 25 35 38.5 65.9 3 2 4 9 9.9 3 33.3 Control-E2 93 4019 20.4 63.4 1 2 3 3.2 0 0.0 Control-E3 98 25 25 25.5 51.0 3 6 5 14 14.36 42.9 Total/Avg 3514 851 911 25.9 (50.1) 192 113 268 573 (16.3) 62(10.8) 0.25 -A1 884 204 222 25.1 48.2 81 36 128 245 27.7 0.25 -A2 434 92100 23.0 44.2 27 14 44 85 19.6 0.25 -A3 1053 230 285 27.1 48.9 83 39 129251 23.8 0.25 -B1 91 33 6 6.6 42.9 0 2 3 5 5.5 2 40.0 0.25 -B2 93 38 2021.5 62.4 4 2 10 16 17.2 10 62.5 0.25 -B3 91 26 16 17.6 46.2 2 3 10 1516.5 6 40.0 0.25 -C1 92 24 34 37.0 63.0 5 3 11 19 20.7 14 73.7 0.25 -C296 31 23 24.0 56.3 1 3 3 7 7.3 5 71.4 0.25 -C3 0.25 -D1 94 25 45 47.974.5 5 9 4 18 19.1 12 66.7 0.25 -D2 99 36 20 20.2 56.6 7 6 6 19 19.2 631.6 0.25 -D3 99 17 46 46.5 63.6 5 7 8 20 20.2 9 45.0 0.25 -E1 97 25 3940.2 66.0 6 9 5 20 20.6 8 40.0 0.25 -E2 97 34 30 30.9 66.0 5 6 5 16 16.54 25.0 0.25 -E3 97 23 33 34.0 57.7 6 7 6 19 19.6 9 47.4 Total/Avg 3417838 919 26.9 (51.4) 237 146 372 755 (22.1) 85 (11.3)

Example 8

In an additional experiment, post-thaw motility and progressive motilitywere checked using CASA at 0, 3 and 5 hours after thawing sex sortedsemen samples (sorted using flow cytometry) treated with vitamin B12 asthe antioxidant. The OSR was not added to the control samples. The semensamples were derived from two Holstein bulls. 0.25 mg/ml concentrationof vitamin B12 was added to the test samples during the staining step,the collecting step (in the catch fluid of the collecting vessel) and inthe cryoprotectant extender prior to freezing the sample (3 step). Theresults are shown in Table 8 below.

TABLE 8 Post-Thaw Motility (3 step) Total Motile Prog Mot VAP VSL VCLALH BCF STR LIN PIA cells (%) (%) (μm/s) (μm/s) (μm/s) (μm) (Hz) (%) (%)(%) Bull A 0 hr Post-Thaw: Bull A - Control 1884 77 53 107 81 211 9 2376 40 Bull A - Control 1275 74 51 99 75 198 9 24 77 41 90 Average (76)(52) 103 78 205 9 24 77 41 Bull A - 0.25 1074 73 51 89 68 183 8 26 77 40Bull A - 0.25 1519 69 50 101 78 195 8 27 78 43 90 Average (71) (51) 9573 189 8 27 78 42 3 hr Post-Thaw: Bull A - Control 1064 48  3 38 29 76 614 77 40 56 Bull A - Control 683 53  7 44 36 80 5 17 84 46 63 Average(50)  (5) 41 32.5 78 5.5 15.5 80.5 43 59.5 Bull A - 0.25 870 64 22 48 3984 8 14 77 41 86 Bull A - 0.25 978 46  7 41 31 83 8 14 77 38 76 Average(55) (15) 45 35 84 8 14 77 40 81 5 hr Post-Thaw: Bull A - Control 903 40 8 14 11 25 3 7 14 9 52 Bull A - Control 517 15  1 12 10 18 2 10 17 1860 Average  (27.5)   (4.5) 13 10.5 21.5 2.5 8.5 15.5 13.5 56 Bull A -0.25 708 61 26 17 17 23 2 6 13 12 79 Bull A - 0.25 747 34  7 20 16 28 38 14 13 72 Average  (47.5)  (16.5) 18.5 16.5 25.5 2.5 7 13.5 12.5 75.5Bull B 0 hr Post-Thaw: Bull B - Control 1878 73 57 88 73 153 7 27 83 49Bull B - Control 1706 72 54 83 69 142 6 28 84 51 90 Average (73)  (55.5)85.5 71 148 7 28 84 50 Bull B - 0.25 1804 79 58 85 70 145 6 28 83 51Bull B - 0.25 1150 79 54 81 67 143 6 29 84 51 90 Average (79) (56) 83 69144 6 29 84 51 3 hr Post-Thaw: Bull B - Control 666 30 10 39 25 80 8 1666 33 60 Bull B - Control 670 50 12 45 37 85 6 16 82 44 72 Average (50)(11) 42 31 82.5 7 16 74 38.5 66 Bull B - 0.25 928 56 18 50 39 96 6 18 8042 83 Bull B - 0.25 772 42 12 46 37 89 6 18 80 42 82 Average (49) (15)48 38 93 6 18 80 42 82.5 5 hr Post-Thaw: Bull B - Control 539  0  0 4016 52 0 26 40 31 46 Bull B - Control 609  0  0 35 26 44 0 71 75 39 61Average  (0)  (0) 37.5 21 48 0 48.5 57.5 35 53.5 Bull B - 0.25 902 43  441 28 85 7 14 70 34 83 Bull B - 0.25 721 21  0 38 21 70 4 14 57 32 73Average (32)  (2) 39.5 24.5 77.5 5.5 14 63.5 33 78

Example 9

In an additional experiment, motility and progressive motility werechecked at 0 hours and 3 hours after thawing sex sorted semen samples(sorted using flow cytometry) treated with the antioxidant, vitamin B12.The OSR was not added to the control sample. The semen samples werederived from two different breeds of bull, one a Holstein the otherTexas Longhorn. 0.25 mg/ml concentration of vitamin B12 was added to thetest samples either (i) during the staining step (1 step-stain); (ii)the staining step and the collecting step (in the catch fluid of thecollecting vessel) (2 step-stain); (iii) in the staining step, thecollecting step and the freezing step (in the cryoprotectant extenderprior to freezing the sample) (3 step)—Table 9 (A) for 3 hr, and (B) for0 hr; or (iv) only prior to the cryopreservation step (1step-freeze)—Table 9 (C) for 3 hr.

TABLE 9 (A) 3 hr Post-Thaw Motility (3 step; 2 step-stain; 1 step-stain)Total Motile Prog Mot VAP VSL VCL ALH BCF STR LIN PIA cells (%) (%)(μm/s) (μm/s) (μm/s) (μm) (Hz) (%) (%) (%) Bull 1 (TL) 3 hr Post-ThawControl (12)  (3) 42 35 77 3 19 83 46 23 3 step (+ + +) 42 25 55 47 1015 23 84 48 73 2 step-stain (+ + −) 25  9 47 41 85 5 21 87 49 47 1step-stain (+ − −) 37 23 56 47 84 5 16 85 57 52 Bull 2 (Jersey) 3 hrPost-Thaw Control (72) (63) 70 59 108 5 19 85 56 72 3 step (+ + +) 75 7081 74 124 5 22 88 58 80 2 step-stain (+ + −) 57 35 60 50 105 5 19 84 4878 1 step-stain (+ − −) 47 32 59 49 98 5 19 84 51 71 Average Avg -Control (42) (33) 56 47 92 4 19 84 51 48 3 step (+ + +) 58 47 68 60 1125 23 86 53 77 2 step-stain (+ + −) 41 22 53 45 95 5 20 85 48 63 1step-stain (+ − −) 42 28 57 48 91 5 18 85 54 61

TABLE 9 (B) 0 hr Post-Thaw Motility (3 step; 2 step-stain; 1 step-stain)Total Motile Prog Mot VAP VSL VCL ALH BCF STR LIN PIA cells (%) (%)(μm/s) (μm/s) (μm/s) (μm) (Hz) (%) (%) (%) Bull 1 (TL) 0 hr Post-ThawControl  (58.5)  (49.5) 93.5 81.0 161.5 7.0 26.0 86.5 52.5 3 step (+ ++) 76 60 89.5 79.5 154.5 7.0 27.5 89.0 56.5 2 step-stain (+ + −) 57 4896.0 84.0 168.0 7.0 25.5 87.5 52.0 1 step-stain (+ − −)  60.5 55 95.083.5 167.0 7.0 26.5 88.5 52.0 Bull 2 (Jersey) 0 hr Post-Thaw Control(79) (68) 105.0 89.0 179.0 7.0 26.0 86.0 53.0 3 step (+ + +) 70  57.794.5 82.0 165.5 6.5 30.0 86.5 52.0 2 step-stain (+ + −) 74 67 97.0 85.0170.0 7.0 27.0 88.0 52.0 1 step-stain (+ − −) 80 71 104.0 88.0 189.0 8.026.0 85.0 49.0

TABLE 9 (C) 3 hr Post-Thaw Motility (1 step-freeze) Bull 3 Total MotileProg Mot VAP VSL VCL ALH BCF STR LIN PIA 3 hr Post-Thaw Cells (%) (%)(μm/s) (μm/s) (μm/s) (μm) (Hz) (%) (%) (%) Control 418 20 0 38 26 67 1412 63 Control 238 29 0 35 23 53 0 14 59 Avg - Control 656 (24.5) (0)36.5 24.5 60 7 13 61 1 step-freeze (− − +) 384 50 10  43 35 80 5 17 63 1step-freeze (− − +) 385 59 17  48 40 86 5 19 83 1 step-freeze (− − +)210 32 4 41 31 75 6 13 67 1 step-freeze (− − +) 301 45 11  45 33 81 6 1385 Avg - 1 step-frz 1280 (46.5)  (10.5) 44.25 34.75 80.5 5.5 15.5 74.5

Example 10

An additional experiment was conducted to assess the pregnancy rate offemale bovines inseminated with sorted sperm cell samples treated withan antioxidant. Semen samples from two bulls were sex sorted using theprotocol described above. 0.25 mg/ml concentration of vitamin B12 wasadded to the test samples during the staining step, the collecting step(in the catch fluid of the collecting vessel) and the freezing step (3step).

Frozen semen straws containing sex sorted sperm cell samples were thawedusing standard procedures. An artificial insemination (AI) gun waswarmed as needed to approach body temperature of the recipient, and astraw was placed in the barrel of the insemination gun. The sealed endof the straw was cut off and a plastic sheath was placed over the strawand gun for hygienic purposes. The female was previously placed in arestraining shoot for insemination. The gun was threaded through thevagina and cervix and semen distributed in the uterine body. 384 femaleswere inseminated, each inseminated with a single sperm cell dose, andeach dose containing 2.1 million sperm cells in 0.25 ml. Pregnancychecks were made 33-40 days post insemination with an ultrasoundmachine. The results are shown in Table 10 below.

TABLE 10 Pregnancies (3 step) Inseminations Percent Pregnancy Bull AControl 87 32.8 0.25 mg/ml 100 35.6 Bull B Control 99 30.9 0.25 mg/ml 9827.0 Total/Average Control 186 (31.9) Total/Average 0.25 mg/ml 198(31.3)

Example 11

Additional pregnancy field trials were performed using the sex-sortedsemen treated with vitamin B12 at 0.25 mg/ml added to the test samplesduring the staining step, the collecting step and again in the freezingstep (3-step), as done in Example 10 was evaluated another time lookingusing semen from five different Holstein bulls. Each semen sample wassplit into control and vitamin B12 (0.25 mg/ml) treatment groups, andlater inseminated into primiparous recipient heifers. Pregnancy checkswere made 33-40 days post insemination using ultrasound. The results areshows in Table 11 below.

TABLE 11 Pregnancies (3 step) Antioxidant Inseminations PercentPregnancy Bull A Control 27.9 0.25 mg/ml 28.4 Bull B Control 26.1 0.25mg/ml 28.0 Bull C Control 32.8 0.25 mg/ml 35.6 Bull D Control 26.0 0.25mg/ml 30.7 Bull E Control 50.0 0.25 mg/ml 57.0 Total/Average Control 499(32.6) Total/Average 0.25 mg/ml 374 (35.9)

Example 12

The levels of DNA fragmentation were also screened using a DNAfragmentation ‘Halomax for animals’ kit (Halotech DNA, sl, Madrid,Spain) to determine if there were any advantageous effects of using theantioxidants during the staining and processing of sex-sorted sperm. Twodifferent breeds of cattle, Jersey and Holstein, were examined using twodifferent concentrations of antioxidant, 0.25 mg/ml and 0.5 mg/ml ofvitamin B12 with the 3 step protocol adding the same concentration ofOSR during cell staining, in the collection catch fluid and prior tocryopreservation. One of the bulls was used to evaluate the effect ofaddition or omission of OSR at one or more of the sperm sorting steps.Motility and the level of DNA fragmentation were both recorded. Theresults are shown in Table 12 below.

TABLE 12 DNA Fragmentation (1 step-stain, 2 step, 3 step) % Motility %DNA Fragmentation 0 hr 3 hr 0 hr 24 hr 48 hr Bull A (Jersey) Control 5717 1 1 1.7 0.25 mg/ml 72 51 0.3 0.3 0.7  0.5 mg/ml 60 42 0.3 0.7 1.3Bull B (Holstein) Control 62 32 0.3 0.3 0.7 0.25 mg/ml 67 32 0 0.3 0.3 0.5 mg/ml 79 48 0.3 0.3 0.7 Bull C (Jersey) Trial 1 Control 79 72 1 1 13 step (+ + +) 70 75 0 0.3 0.3 2 step-stain (+ + −) 74 57 0.3 1 1 1step-stain (+ − −) 80 47 0.7 1 1.3 Trial 2 Control 59 12 0.3 1.7 1.7 3step (+ + +) 76 42 0.3 0.3 0.7 2 step-stain (+ + −) 57 25 0.3 0.7 0.7 1step-stain (+ − −) 61 37 0.7 1 1

Example 13

The effect of OSR on motility was evaluated as a function of theconcentration of the sex-sorted sperm in the frozen straw. Tests wereperformed using vitamin B12 as the OSR at three different concentrationsof the antioxidant: 0.15 mg/ml; 0.25 mg/ml and 0.35 mg/ml; all wereadded to the test samples during the staining step, the collecting stepand again in the freezing step (3-step), as done in Example 8. Holsteinsperm was evaluated at three sperm cell concentrations based upon totalnumber of sperm per straw: 1 million sperm/straw; 2.1 millionsperm/straw; and 5 million sperm/straw. Motility was recorded 3 hrpost-thaw.

A Jersey sperm sample was also evaluated in the same manner using 0.15mg/ml or 0.25 mg/ml vitamin B12 at each of the three stages (3 step),but only at the 2.1 million sperm/straw concentration. The results areshown in Table 13.

TABLE 13 Frozen Sperm Cell Concentration (3 step) Motile Prog VAP VSLVCL ALH BCF STR LIN PIA (Holstein) 1M/straw Control 20 4 40 32 76 3 1578 42 43 .15 mg/ml 48 18 48 42 81 4 12 88 53 66 .25 mg/ml 48 29 55 47 924 22 87 53 58 .35 mg/ml 65 46 58 51 94 4 23 88 55 70 2.1M/straw Control28 2 39 33 73 6 16 85 46 41 .15 mg/ml 49 10 44 38 79 5 18 86 48 72 .25mg/ml 48 18 46 40 81 5 20 87 50 76 .35 mg/ml 46 30 58 51 99 5 23 88 5360 5M/straw Control 20 1 39 29 69 7 13 74 43 46 .25 mg/ml 24 2 41 28 697 14 70 42 68 .35 mg/ml 28 3 41 32 68 7 14 69 43 69  .5 mg/ml 14 3 43 3071 6 15 70 43 50 (Jersey) 2.1M/straw Control 25 2 37 28 55 2 11 76 52 53.15 mg/ml 25 4 42 35 75 6 15 84 47 63 .25 mg/ml 48 20 51 43 87 5 20 8550 78

Example 14

The effect of a different OSR on bovine sperm motility was evaluatedafter zero hour and a three hour post-thaw period. The OSR,α-tocopheryl, a form of Vitamin E, was purchased aspolyoxyethanyl-α-tocopheryl sebacate′ in a 15% stock solution (Aldrich).Tests were performed using α-tocopheryl as the OSR, at three differentconcentrations: 0.01 mg/ml; 0.1 mg/ml and 0.5 mg/ml; all were added tothe test samples during the staining step, the collecting step and againin the freezing step (3-step), as done in the prior Examples. Sex-sortedHolstein sperm was evaluated using a standard sperm concentration of 2.1million sperm/straw processed from three separate bulls. Motility of thetreated samples were compared to untreated controls and recorded at 0 hrand 3 hr post-thaw.

TABLE 14 Motility with α-tocopheryl (vitamin E) (3 step) Total cellsMotile Prog VAP VSL VCL ALH BCF STR LIN Holstein 0 hr Post-Thaw Bull AControl 548 56 25 75 52 151 7 22 74 39 0.01 617 72 17 100 49 201 10 2056 27 0.1 458 73 33 99 62 217 9 22 66 32 0.5 424 63 36 97 68 214 9 22 7234 Bull B Control 548 56 25 75 52 151 7 22 74 39 0.01 617 72 17 100 49201 10 20 56 27 0.1 458 73 33 99 62 217 9 22 66 32 0.5 424 63 36 97 68214 9 22 72 34 Bull C (1) Control 280 50 30 66 51 140 7 20 78 39 0.01530 59 39 89 68 188 10 20 76 38 0.1 518 63 35 86 61 196 10 20 72 33 0.5269 30 17 75 53 179 8 22 72 31 Bull C (2) Control 364 23 12 65 54 116 525 82 49 0.01 347 36 27 86 78 140 6 26 90 57 0.1 822 27 18 84 75 149 625 88 52 0.5 764 29 18 74 64 130 7 28 87 55 Holstein 3 hr Post-ThawA-toc Bull A Control 260 25 6 39 33 71 5 16 83 47 0.01 253 64 20 51 4292 5 21 84 49 0.1 297 62 32 54 46 101 5 22 86 48 0.5 235 54 29 60 51 1065 24 85 49 Bull B Control 309 29 8 46 37 83 6 19 82 46 0.01 126 28 9 5439 103 4 21 76 41 0.1 154 14 4 56 36 100 6 18 73 38 0.5 151 36 10 57 41104 5 22 77 44 Bull C (1) Control 200 14 2 40 33 77 5 18 83 44 0.01 22219 3 41 38 60 4 29 93 70 0.1 155 15 5 49 41 94 7 21 84 45 0.5 82 26 1350 44 89 5 18 87 50 Bull C (2) Control 207 2 0 19 15 26 1 11 73 27 0.01270 6 0 35 21 55 0 13 60 39 0.1 658 4 0 38 29 63 1 8 76 46 0.5 278 2 037 21 64 0 14 55 33

Example 15

The effect of a third OSR on bovine sperm motility was evaluated afterzero hour, three hour and six hour post-thaw periods.Alpha-ketoglutarate (AKG) was freshly made and used at three differentconcentrations: 0.25 mg/ml; 0.35 mg/ml and 0.45 mg/ml; all were added tothe test samples during the staining step, the collecting step and againin the freezing step (3-step), as done in the prior Examples. Sex-sortedHolstein and Jersey bovine sperm were evaluated using a standardconcentration of 2.1 M sperm/straw. Motility of the treated samples wascompared to untreated controls at 0 hr, 3 hr and 6 hr post-thaw.

TABLE 15 Alpha-keto Glutarate (AKG) (3 step) Motile Prog VAP VSL VCL ALHBCF STR LIN PIA Holstein-0 hr control 41 16 69 44 143 8 21 66 34 .25mg/ml 91 75 104 81 185 8 27 82 48 .35 mg/ml 80 65 94 80 165 7 29 85 51.45 mg/ml 86 75 98 88 162 7 30 89 57 Holstein - 3 hr control 4 0 36 2453 0 10 67 48 44 .25 mg/ml 77 60 99 79 176 7 26 81 45 89 .35 mg/ml 63 4885 70 145 6 25 83 50 80 .45 mg/ml 60 56 94 80 145 5 24 85 56 80 control30 7 40 30 68 3 13 74 46 44 .25 mg/ml 71 55 96 77 169 6 26 81 47 89 .35mg/ml 64 38 69 57 117 5 21 84 50 80 .45 mg/ml 60 50 90 75 144 6 25 84 5380 Holstein - 6 hr control 4 0 38 23 55 0 12 61 42 56 .25 mg/ml 36 7 4433 87 6 17 76 39 73 .35 mg/ml 58 7 42 32 80 7 16 76 40 85 .45 mg/ml 5222 55 46 96 5 22 82 48 79 control 4 0 38 23 55 0 12 61 42 56 .25 mg/ml36 7 44 33 87 6 17 76 39 73 .35 mg/ml 58 7 42 32 80 7 16 76 40 85 .45mg/ml 52 22 55 46 96 5 22 82 48 79 Jersey - 0 hr control 72 60 82 72 1416 25 87 53 .25 mg/ml 67 51 83 68 152 7 24 83 48 .35 mg/ml 57 43 79 63146 6 25 82 47 .45 mg/ml 81 66 81 70 133 6 27 87 56 Jersey - 3 hrcontrol 13 1 36 24 65 2 14 66 38 42 .25 mg/ml 33 16 65 49 117 5 24 79 4662 .35 mg/ml 26 10 59 46 110 6 25 80 45 54 .45 mg/ml 59 25 67 50 66 6 2578 45 71

Example 16

The effect of combining two antioxidants on bovine sperm motility wasevaluated after zero hour, three hour and six hour post-thaw periods.Vitamin B12 was used at 0.25 mg/ml and fresh alpha-ketoglutarate (AKG)was used at 0.35 mg/ml. Either vitamin B12 or AKG or both treatmentswere added at the designated concentrations during the staining step,the collecting step and again in the freezing step (3-step), as doneearlier. Sex-sorted sperm from three Jersey bulls were evaluated usingstandard 2.1 M sperm/straw. Motility of the treated samples was comparedto untreated controls at 0 hr, 3 hr and 6 hr post-thaw.

TABLE 16 Combination AKG, Vitamin B12, and Combination (3 step) MotilePROG VAP VSL VCL ALH BCF STR LIN Jersey 1 - 0 hr Control 75 67 99 87 1626 30 88 56 B12 - 0.25 mg/ml 88 78 97 87 157 6 33 89 58 AKG - 0.35 mg/ml87 75 93 81 148 6 31 88 57 COMBO 78 69 93 83 149 6 32 89 57 Jersey 1 - 3hr Control 43 5 41 33 74 6 15 56 46 B12 - 0.25 mg/ml 53 34 58 51 93 4 2387 55 AKG - 0.35 mg/ml 60 44 82 67 136 5 27 83 50 COMBO 51 36 79 63 1295 25 80 50 Jersey 1 - 6 hr Control 2 1 48 38 58 3 8 75 64 B12 - 0.25mg/ml 37 2 38 28 61 4 15 73 48 AKG - 0.35 mg/ml 36 18 50 42 90 4 22 8246 COMBO 35 6 45 34 81 5 17 76 43 Jersey 2 - 3 hr Control 18 1 35 22 575 13 61 38 B12 - 0.25 mg/ml 66 30 51 43 93 5 18 84 47 AKG - 0.35 mg/ml64 50 78 62 117 5 19 82 56 COMBO 50 38 72 57 124 5 24 80 48 Jersey 2 - 6hr Control 1 0 16 16 35 0 0 50 23 B12 - 0.25 mg/ml 24 1 36 24 51 1 17 6547 AKG - 0.35 mg/ml 44 5 42 36 68 2 17 86 54 COMBO 46 18 61 46 111 5 2177 44 Jersey 3 - 0 hr Control 80 60 100 80 186 8 26 81 46 B12 - 0.25mg/ml 83 70 89 76 160 7 27 85 51 AKG - 0.35 mg/ml 86 73 95 82 170 7 2785 50 COMBO 89 74 99 81 177 7 28 84 49 Jersey 3 - 3 hr Control 64 22 4738 88 6 16 81 45 B12 - 0.25 mg/ml 73 52 64 52 104 10 20 81 51 AKG - 0.35mg/ml 80 44 69 53 118 6 19 79 47 COMBO 77 30 56 44 107 7 19 79 42 Jersey3 - 6 hr Control 16 0.5 38 24 54 1 17 64 47 B12 - 0.25 mg/ml 44 1 37 2456 3 14 66 44 AKG - 0.35 mg/ml 79 11 46 33 84 6 15 74 41 COMBO 72 12 4934 98 8 16 71 35

Example 17

In an additional experiment, motility and progressive motility of femaleand male sex-sorted Deer sperm were checked at 0, 1 and 3 hours afterthawing the sex sorted semen samples that were treated with theantioxidant, vitamin B12 at two concentrations: 0.25 mg/ml and 0.35mg/ml; all samples were treated during staining, catch and prior tocryopreservation (3 step). The OSR was not added to the control sample.The semen samples were derived from two white tailed bucks. Each samplewas sorted for both male and female enriched populations of sperm.

TABLE 17 Deer (male and female) - Vitamin B12 (3 step) TOTAL MOTILE PROGVAP VSL VCL ALH BCF STR 0 HR FEMALE Buck A CONTROL 1887 74 53 85 67 1577 24 78 0.25 2803 82 53 83 62 157 7 24 74 0.35 2484 84 55 92 68 169 7 2474 MALE CONTROL 2939 81 55 94 70 178 7 24 76 0.25 3036 76 49 91 66 173 724 73 0.35 2448 86 54 98 71 181 8 24 73 FEMALE Buck B CONTROL 2025 80 3692 56 185 8 22 64 0.25 1531 82 40 91 56 179 7 22 65 0.35 1859 89 43 10569 198 8 23 66 MALE CONTROL 2114 85 39 103 62 202 8 22 63 0.25 1169 8135 109 63 219 8 23 61 0.35 1658 89 46 103 67 197 8 23 66 1 HR FEMALEBuck A CONTROL 1286 64 29 86 57 170 8 21 68 0.25 1380 78 41 97 64 185 821 67 0.35 1808 80 31 109 68 216 9 22 63 MALE CONTROL 1612 67 27 71 50141 7 22 73 0.25 1817 57 23 64 45 125 7 22 70 0.35 3169 85 52 105 75 1988 23 72 FEMALE Buck B CONTROL 1371 81 26 65 43 127 6 19 68 0.25 1541 8328 84 51 158 8 19 64 0.35 2637 92 32 116 66 214 9 20 58 MALE CONTROL1520 84 48 86 57 169 6 23 70 0.25 1086 84 46 104 66 203 8 22 67 0.351881 92 46 112 71 216 8 22 65 2 HR FEMALE Buck A CONTROL 1188 58 16 5842 114 7 18 74 0.25 1045 75 40 97 65 190 9 20 68 0.35 877 54 19 68 50137 8 20 75 MALE CONTROL 1325 59 13 51 38 101 6 19 77 0.25 1523 56 24 6646 130 6 22 70 0.35 1797 58 39 101 75 193 8 23 76 FEMALE Buck B CONTROL1125 73 15 56 38 111 7 18 69 0.25 834 64 11 53 36 106 7 16 68 0.35 137885 26 100 57 190 8 19 60 MALE CONTROL 1603 77 37 66 46 126 6 19 73 0.25866 78 47 95 67 111 7 21 71 0.35 1760 89 41 78 51 149 7 20 70

Example 18

In an another experiment, the progressive motility of conventional(i.e., unsorted) semen was compared to conventional semen treated withvitamin B12 at a concentration of 0.25 mg/ml and AKG at a concentrationof 0.35 mg/ml (the “anti-oxidant treatment”) at 0, 1 and 3 hourspost-thaw.

Semen from 1159 bulls was obtained. Raw ejaculates were diluted 1:1 witha Tris-based medium containing 20% egg yolk. The Tris-based medium usedwith the treatment group also contained the antioxidant treatment, whilethe Tris-based medium used with the control group did not. Ejaculateswere cooled to 4° C. After 90 minutes a glycerol-based cryoextender wasadded to the ejaculates. The glycerol-based cryoextender used with thetreatment group also contained the anti-oxidant treatment, while theglycerol-based cryoextender used with the control group did not.Ejaculates were allowed to sit for at least 2 hours before being placedinto artificial insemination straws and then frozen in liquid nitrogen.Straws were thawed and post-thaw motility of the ejaculates was checkedat 0 and 3 hours.

0 hour post-thaw motility measured 61.08 (SE=0.48) for the control groupand 65.13 (SE=0.76) for the treatment group. 3 hour post-thaw motilitymeasured 49.85 (SE=0.77) for the control group and 59.78 (SE=1.16) forthe treatment group. Convergence (3 hour post-thaw motility/0 hourpost-thaw motility) for the control group measured 0.80 (SE=0.01) and0.92 (SE=0.01) for the treatment group.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. As such, theparticular embodiments, elements, terms, or expressions disclosed by thedescription, or shown in the figures accompanying this application arenot intended to be limiting, but rather are examples of the numerous andvaried embodiments generically encompassed by the invention or itsequivalents with respect to any particular element thereof. In addition,the specific description of a single embodiment or element of theinvention may not explicitly describe all embodiments or elementspossible; many alternatives are implicitly disclosed by the descriptionand figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As example, itshould be understood that all steps of a method may be disclosed as anaction, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As another example, the disclosure of a “sorter” should beunderstood to encompass disclosure of the act of “sorting,” whetherexplicitly discussed or not, and conversely, effective disclosure of theact of “sorting” should be understood to encompass disclosure of a“sorter.” Such alternative terms for each element or step are to beunderstood to be explicitly included in the description.

In addition, it should be understood that unless utilization of aspecific term in this application is inconsistent with common use andinterpretation of that term, dictionary definitions should be understoodto be included in the description for each term as contained in theRandom House Webster's Unabridged Dictionary, second edition, eachdefinition hereby incorporated by reference.

Moreover, for the purposes of the present invention, the term “a” or“an” before an item also refers to one or more of that item; forexample, “a container” refers to one or more of the containers. As such,the terms “a” or “an”, “one or more” and “at least one” can be usedinterchangeably herein. Further, as used herein the term “or” means“and/or” unless specifically indicated otherwise.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the present invention, and the terms used in those earlierdocuments which may be similarly used in this disclosure, shall notalter the intended definition of those same terms as defined or intendedherein.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of the description of the currentinvention, and the applicant expressly reserves the right to use all ora portion of such incorporated content as additional description tosupport any or all of the claims or any element or component thereof.The applicant further expressly reserves the right to move any portionor all of the incorporated content of such claims or any element orcomponent thereof from the description into the claims, or vice versa,as necessary to define the invention for which protection is sought bythis application or by any subsequent application or continuation,division, or continuation-in-part application thereof, or to obtain anybenefit for reduction in fees in compliance with relevant patent laws,rules, or regulations of any country or treaty, and such incorporatecontent shall survive the entire pendency of this application as well asany subsequent continuation, division, continuation-in-part applicationfilings or any reissue or extension thereof.

The claims set forth in this specification are intended to describe themetes and bounds of a limited number of the preferred embodiments of theinvention and are not to be construed as the broadest embodiment of theinvention or a complete listing of embodiments of the invention that maybe claimed. The applicant does not waive any right to develop furtherclaims based upon the description set forth above as a part of anycontinuation, division, or continuation-in-part, or similar application.

1. A sperm cell composition comprising a sperm cell sample and vitaminB12, or a vitamin B12 vitamer, at a concentration of 0.01 mg/ml to 1mg/ml.
 2. The sperm cell composition of claim 1, further comprisingalpha ketoglutarate at a concentration of 0.01 mg/ml to 5 mg/ml.
 3. Thesperm cell composition of claim 2, wherein the concentration of theadded alpha ketoglutarate is selected from the group consisting of: 0.01to 5.0 mg/ml; 0.01 to 0.25 mg/ml; 0.01 to 0.5 mg/ml; 0.01 to 1 mg/ml;0.01 to 2.5 mg/ml; 0.01 to 5 mg/ml; 0.05 to 0.1 mg/ml; 0.05 to 1.0mg/ml; 0.05 to 2.5 mg/ml; 0.1 to 0.25 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 1mg/ml; 0.1 to 2.5 mg/ml; 0.1 to 5 mg/ml; 0.15 to 0.45 mg/ml; 0.15 to 0.5mg/ml; 0.25 to 0.35 mg/ml; 0.25 to 0.5 mg/ml; 0.25 to 1 mg/ml; 0.25 to2.5 mg/ml; 0.25 to 5 mg/ml; 0.35 to 0.5 mg/ml; 0.35 to 1 mg/ml; 0.35 to2.5 mg/ml; 0.35 to 5 mg/ml; 0.5 to 1 mg/ml; 0.5 to 2.5 mg/ml; 0.5 to 5mg/ml; 1 to 2.5 mg/ml; and 1 to 5 mg/ml.
 4. The sperm cell compositionof claim 2, wherein the concentration of the vitamin B12, or vitamin B12vitamer, or alpha ketoglutarate, is selected from the group consistingof: 0.05 mg/ml; 0.1 mg/ml; 0.15 mg/ml; 0.25 mg/ml; 0.35 mg/ml; 0.45mg/ml; and 0.5 mg/ml.
 5. The sperm cell composition of claim 2, whereinthe concentration of the vitamin B12, or vitamin B12 vitamer, or alphaketoglutarate, is selected from the group consisting of: 0.15 mg/ml;0.25 mg/ml; and 0.35 mg/ml.
 6. The sperm cell composition of claim 2,wherein the sperm cell composition is cryopreserved.
 7. The sperm cellcomposition of claim 2, wherein the sperm cell sample is derived from ahuman, bovine, swine, ovine, equine, deer, elk, buffalo, canine, feline,chimpanzee or gorilla, or whale, dolphin or other marine mammal.